Role of inflammation, oxidative stress, and autonomic nervous system activation during the development of right and left cardiac remodeling in experimental pulmonary arterial hypertension.

This study investigated the impact of experimental pulmonary arterial hypertension (PAH) progression by evaluating morphometric and functional parameters, oxidative stress, autonomic nervous system (ANS) activation, and inflammation in the right (RV) and left (LV) ventricles. Male rats were first divided into two groups: monocrotaline (MCT) and control. The MCT group received a single MCT injection (60 mg/kg, intraperitoneal), while control received saline. The MCT and control groups were further divided into four cohorts based on how long they were observed: 1, 2, 3, and 4 weeks. Animals were submitted to echocardiographic and hemodynamic analysis. RV and LV were used for morphometric, biochemical, and histological measurements. Autonomic modulation was evaluated by cardiac spectral analysis, considering two components: low frequency (LF) and high frequency (HF). Lung and liver weight was used for morphometric analysis. MCT induced 100% mortality at 4 weeks. In the RV, disease progression led to mild inflammation and enhanced reactive oxygen species (ROS) in week 1, followed by moderate inflammation, ROS production, and hypertrophy in week 2. By week 3, there was moderate inflammation, oxidative stress, and ANS imbalance, with development of right heart dysfunction. LV biochemical changes and inflammation were observed at week 3. The initial changes appeared to be related to inflammation and ROS, and the later ones to inflammation, oxidative stress, and ANS imbalance in MCT animals. This study reinforces the severity of the disease in the RV, the late effects in the LV, and the role of ANS imbalance in the development of heart dysfunction.

Development of an in vitro model to study tooth cystogenesis.

AIM: To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. METHODOLOGY: To form spheroid structures, various numbers (1 × 104 , 5 × 104 or 1 × 105 ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis. RESULTS: Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. CONCLUSIONS: This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.

Olive oil and prevention of chronic diseases: Summary of an International conference.

Olive oil is the foremost source of fat in the Mediterranean area and, among other features, sets the Mediterranean diet apart from other dietary regimens. In January 2018, the International Olive Council convened several worldwide experts at the Robert Mondavi Institute (Davis, CA), to discuss and summarize the available data on the effects of olive oil consumption on human health. In this paper, we critically provide a synthesis of the main reported findings, which underscore how and why consuming this oil as part of a balanced diet and healthful lifestyle improves prognosis and extends life- and health-spans.

Epithelial oral mucosal cells: Do they behave differently when exposed to oral carcinogens?

OBJECTIVE: To assess the level of maturation and proliferation of epithelial cells and the correlation with immunocytochemical expression of adhesion (E-cadherin) and cell differentiation (involucrin) markers. METHODS: Cytopathological samples were obtained from four groups of patients: control (CG, n=30); alcohol/tobacco (ATG, n=31), leucoplakia (LG, n=31), and squamous cell carcinoma (SCCG, n=22). Cytopathological smears were collected from all groups for AgNOR, Papanicolaou and immunocytochemical staining. RESULTS: There was an increase in anucleated cells in ATG compared to CG and in LG compared to lesion-free groups (P<.05). In addition, there was a higher rate of intermediate cells in lesion-free groups than in LG (P=.001). When these findings were correlated with positive E-cadherin expression, there was a smaller number of anucleated and intermediate cells (P<.05). The proliferation rate was higher in the SCCG than in the CG (P<.05) and in the ATG compared to LG (P<.05). Moreover, cell proliferation increased in the presence of positive E-cadherin expression in the ATG and LG. No statistically significant results were obtained for involucrin analysis. CONCLUSION: Cytopathology combined with quantitative techniques such as Papanicolaou, AgNOR, and immunocytochemical expression of E-cadherin detects changes associated with oral carcinogenesis. The innovative approach used in this study allows assessing the expression of cell adhesion (E-cadherin) and differentiation (involucrin) markers by means of oral mucosal cytopathology. The E-cadherin imunocytochemical expression indicated changes associated with the oral carcinogenesis process. An increase in cell proliferation rate in oral squamous cell carcinoma group was associated with the lower immunoexpression of E-cadherin. Cytopathology combined with quantitative techniques and immunocytochemical expression of E-cadherin may detect early alterations associated with oral carcinogenesis.

Circulating microRNAs in Huntington's disease: Emerging mediators in metabolic impairment.

Huntington's disease (HD) is a hereditary neurodegenerative disease, with peripheral consequences that negatively contribute to quality of life. Circulating microRNAs (cmiRNAs) are being explored for their roles in intercellular communication and gene expression regulation, which allows gaining insight into the regulation of crosstalk between neuronal and peripheral tissues. Here, we explore the cmiRNA profile of plasma samples from fifteen symptomatic patients, with 40-45 CAG repeats in the HTT gene, and seven healthy matched controls. Isolated miRNAs from plasma samples were run against human miRNome panels, which have sequences for 752 human mature miRNAs. We found that 168 cmiRNAs are altered in symptomatic patients. Considering Bonferroni's correction, miR-877-5p, miR-223-3p, miR-223-5p, miR-30d-5p, miR-128, miR-22-5p, miR-222-3p, miR-338-3p, miR-130b-3p, miR-425-5p, miR-628-3p, miR-361-5p, miR-942 are significantly increased in HD patients as compared with controls. Moreover, after patient's organization according to approved HD scales, miR-122-5p is significantly decreased in HD patients with Unified Huntington's Disease Rating Scale >24, whereas an increase in miR-100-5p levels and a decrease in miR-641 and miR-330-3p levels were recorded when patients were rearranged by Total Functional Capacity. These results suggest that cmiRNA profile could be further modified by disease progression, making cmiRNAs useful as monitoring biomarkers. Analysis of target genes indicated a general overexpression of cmiRNAs implicated in metabolism regulation. Profiling cmiRNA of HD subjects opens the possibility of personalized therapies for different groups of HD patients, based on disease modifiers: regulation of altered pathways might contribute to not only alleviate disease symptoms, but also influence HD progression.

Responsiveness of clinical outcome measures in Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease (CMT) is a very slowly progressive neuropathy which makes it difficult to detect disease progression over time and to assess intervention efficacy. Experience from completed clinical trials with ascorbic acid and natural history studies confirm difficulties in detecting such changes. Consequently, sensitive-to-change outcome measures (OMs) are urgently needed. METHODS: The relative responsiveness of clinical scales of the Italian-UK ascorbic acid trial (placebo arm) were assessed by using the standardized response mean (SRM), which is the ratio of the paired scores mean change over time to the standard deviation of the score change (0 is worst responsiveness). RESULTS: Little worsening of OM scores was found over 2 years. In detail, the primary OM of the trial, the CMT Neuropathy Score version 1 (CMTNSv1), showed low responsiveness (SRM 0.13). Some CMTNS items showed slightly greater responsiveness (CMT Examination Score 0.17; CMTNS Signs 0.19). Myometric assessments of handgrip and foot dorsiflexion strength were the most responsive (SRM -0.31 and -0.38, respectively). Amongst the other measures, the nine-hole peg test, which assesses upper limb functioning, showed the best sensitivity to change (SRM 0.28). CONCLUSIONS: Overall these OMs showed low or negligible responsiveness, confirming the need to improve current OMs and to develop novel ones for prognostic and interventional studies. However, handgrip and foot dorsiflexion myometry are worth retaining for future trials as they were the most responsive and are likely to be clinically relevant for patients.

Argan oil reduces, in rats, the high fat diet-induced metabolic effects of obesity.

BACKGROUND AND AIM: Obesity is a multi-factorial disorder which is of worldwide concern. In addition to calorie control, some specific dietary components might help resolving some of the complication of obesity, by providing antioxidant and anti-inflammatory activities. We investigated the effect of argan oil supplementation on plasma lipid profile and oxidant-antioxidant status of rats with high-fat diet (HFD)-induced obesity compared with rats fed a normal diet (ND). METHODS AND RESULTS: We used an animal model of high fat diet-induced obesity to study the metabolic effects of argan oil and we measured several markers lipid and redox statuses. Consumption of a high-fat diet led to an increase in serum total cholesterol (TC), LDL-cholesterol (LDL-C), and triacylglycerols (TAG) concentrations; however, argan oil blunted the increases of TC, LDL-C and TG, glucose, and insulin. Plasma total antioxidant capacity, erythrocyte catalase and superoxide dismutase activities were lower, whereas plasma hydroperoxide, thiobarbituric acid-reacting substances, and susceptibility of LDL to copper-induced oxidation were higher in obese rats compared with normal rats. Administration of argan oil ameliorated all these indices of redox status. CONCLUSIONS: Proper diet and lifestyle should be foremost implemented to reduce the lipoprotein metabolism and oxidant/antioxidant status alterations brought about by obesity. In addition, argan oil reduces the metabolic effects of obesity and its use might be promoted within the context of a balanced diet.

Is overwork weakness relevant in Charcot-Marie-Tooth disease?

BACKGROUND: In overwork weakness (OW), muscles are increasingly weakened by exercise, work or daily activities. Although it is a well-established phenomenon in several neuromuscular disorders, it is debated whether it occurs in Charcot-Marie-Tooth disease (CMT). Dominant limb muscles undergo a heavier overload than non-dominant and therefore if OW occurs we would expect them to become weaker. Four previous studies, comparing dominant and non-dominant hand strength in CMT series employing manual testing or myometry, gave contradictory results. Moreover, none of them examined the behaviour of lower limb muscles. METHODS: We tested the OW hypothesis in 271 CMT1A adult patients by comparing bilateral intrinsic hand and leg muscle strength with manual testing as well as manual dexterity. RESULTS: We found no significant difference between sides for the strength of first dorsal interosseous, abductor pollicis brevis, anterior tibialis and triceps surae. Dominant side muscles did not become weaker than non-dominant with increasing age and disease severity (assessed with the CMT Neuropathy Score); in fact, the dominant triceps surae was slightly stronger than the non-dominant with increasing age and disease severity. DISCUSSION: Our data does not support the OW hypothesis and the consequent harmful effect of exercise in patients with CMT1A. Physical activity should be encouraged, and rehabilitation remains the most effective treatment for CMT patients.

Chronic hydroxytyrosol feeding modulates glutathione-mediated oxido-reduction pathways in adipose tissue: a nutrigenomic study.

BACKGROUND AND AIM: Hydroxytyrosol (HT) is the most prominent phenolic component of olives, olive oil, and their by-products, e.g. olive mill waste water. As the link between HT consumption (via extra virgin olive oil intake) and better cardiovascular prognosis is being scientifically validated, HT is entering the market as a potentially useful supplement for cardiovascular disease prevention. One of the target organs in cardiometabolic prevention is the adipose tissue, where inflammation, oxidative stress, and secretion of adipocytokines contribute to cardiovascular risk. METHODS AND RESULTS: We explored the nutrigenomic effects of long-term supplementation with nutritionally-relevant doses of HT, i.e. 0.03 gm% - with specific reference to the adipose tissue and glutathione metabolism - and we explored underlying mechanisms in vitro. We show that HT modulates the antioxidant network in the adipose tissue, as mediated by glutathione (GSH) and associated enzymes. We also confirmed the GSH-modulating activities of HT in cultured adipocytes, where low, physiological HT concentrations were able to blunt the H2O2-induced GSH/GSSG alteration indicative of oxidative stress. In terms of surrogate markers of cardiovascular disease, we recorded significantly decreased circulating leptin concentrations in mice fed with HT as compared with controls. CONCLUSIONS: HT - in nutritionally relevant amounts - is able to positively modulate the glutathione-driven antioxidant enzymatic machinery in the adipose tissue. Because HT is generally recognized as safe (GRAS) and exhibits an excellent safety profile in vitro and in vivo, its future employment as adjunct treatment of metabolic syndrome can be envisioned, pending specific trials.

Moderate alcohol use and health: a consensus document.

AIMS: The aim of this consensus paper is to review the available evidence on the association between moderate alcohol use, health and disease and to provide a working document to the scientific and health professional communities. DATA SYNTHESIS: In healthy adults and in the elderly, spontaneous consumption of alcoholic beverages within 30 g ethanol/d for men and 15 g/d for women is to be considered acceptable and do not deserve intervention by the primary care physician or the health professional in charge. Patients with increased risk for specific diseases, for example, women with familiar history of breast cancer, or subjects with familiar history of early cardiovascular disease, or cardiovascular patients should discuss with their physician their drinking habits. No abstainer should be advised to drink for health reasons. Alcohol use must be discouraged in specific physiological or personal situations or in selected age classes (children and adolescents, pregnant and lactating women and recovering alcoholics). Moreover, the possible interactions between alcohol and acute or chronic drug use must be discussed with the primary care physician. CONCLUSIONS: The choice to consume alcohol should be based on individual considerations, taking into account the influence on health and diet, the risk of alcoholism and abuse, the effect on behaviour and other factors that may vary with age and lifestyle. Moderation in drinking and development of an associated lifestyle culture should be fostered.

TLR4 expression and functionality are downregulated in glioblastoma cells and in tumor-associated macrophages: A new mechanism of immune evasion?

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.

Olive oil and health: summary of the II international conference on olive oil and health consensus report, Jaén and Córdoba (Spain) 2008.

Olive oil (OO) is the most representative food of the traditional Mediterranean Diet (MedDiet). Increasing evidence suggests that monounsaturated fatty acids (MUFA) as a nutrient, OO as a food, and the MedDiet as a food pattern are associated with a decreased risk of cardiovascular disease, obesity, metabolic syndrome, type 2 diabetes and hypertension. A MedDiet rich in OO and OO per se has been shown to improve cardiovascular risk factors, such as lipid profiles, blood pressure, postprandial hyperlipidemia, endothelial dysfunction, oxidative stress, and antithrombotic profiles. Some of these beneficial effects can be attributed to the OO minor components. Therefore, the definition of the MedDiet should include OO. Phenolic compounds in OO have shown antioxidant and anti-inflammatory properties, prevent lipoperoxidation, induce favorable changes of lipid profile, improve endothelial function, and disclose antithrombotic properties. Observational studies from Mediterranean cohorts have suggested that dietary MUFA may be protective against age-related cognitive decline and Alzheimer's disease. Recent studies consistently support the concept that the OO-rich MedDiet is compatible with healthier aging and increased longevity. In countries where the population adheres to the MedDiet, such as Spain, Greece and Italy, and OO is the principal source of fat, rates of cancer incidence are lower than in northern European countries. Experimental and human cellular studies have provided new evidence on the potential protective effect of OO on cancer. Furthermore, results of case-control and cohort studies suggest that MUFA intake including OO is associated with a reduction in cancer risk (mainly breast, colorectal and prostate cancers).

Lipoic acid significantly restores, in rats, the age-related decline in vasomotion.

BACKGROUND AND PURPOSE: The age-related decline in vasorelaxation is largely due to ceramide-induced induction of phosphatase 2A (PP2A), which limits nitric oxide synthase (eNOS) phosphorylation at stimulatory sites. We hypothesized that ceramide accumulation was from an age-related loss of endothelial glutathione (GSH) and subsequent activation of neutral sphingomyelinase (nSMase), an enzyme whose activity increases when GSH is limited. EXPERIMENTAL APPROACH: Old (30-32 mo) F344xBN rats were given (R)-alpha-lipoic acid (LA), an agent known to induce GSH synthesis. Vasorelaxation was measured in aortic rings; GSH and ceramide levels, activity of nSMase and eNOS phosphorylation (by Western blot) was measured in aortic endothelial cells, isolated from the same aortas. KEY RESULTS: In old animals, endothelium-dependent relaxation in aortic rings was decreased, GSH levels and its redox state in aortic endothelia were over 30% lower and nSMase activity and endothelial ceramide levels were three-fold increased, relative to young (2-4 mo) rats. LA treatment of old animals improved relaxation in aortic rings, reversed the changes in endothelial GSH, in nSMase activities and in ceramide levels. Similar effects on GSH levels and nSMase activity in old rats were also induced by treatment with GSH monoethylester. Activation (by phosphorylation) of eNOS was decreased by about 50% in old rats and this age-related decrease was partially reversed by LA treatment. CONCLUSIONS AND IMPLICATIONS: Decreased endothelial GSH was partly responsible for the age-related loss of vascular endothelial function and LA might be therapeutically evaluated to treat endothelial dysfunction.

Autophagy analysis in oral carcinogenesis.

OBJECTIVE: The aim of this study was to evaluate the levels of autophagy in oral leukoplakia and squamous cell carcinoma and to correlate with clinical pathological features, as well as, the evolution of these lesions. METHODOLOGY: 7 Normal oral mucosa, 51 oral leukoplakias, and 120 oral squamous cell carcinomas (OSCC) were included in the study. Histological sections of the mucosa and leukoplakias were evaluated throughout their length, while the carcinomas were evaluated using Tissue Microarray. After the immunohistochemical technique, LC3-II positive cells were quantified in the different epithelial layers of the mucosa and leukoplakias and in the microarrays of the squamous cell carcinomas. The correlation between positive cells with the different clinical-pathological variables and with the evolution of the lesions was tested using the t test, ANOVA, and Kaplan-Meier survival analysis. RESULTS: We observed increased levels of autophagy in the oral squamous cell carcinomas (p<0.001) in relation to the other groups, but without any association with poorer evolution or survival of these patients. Among the leukoplakias, we observed a higher percentage of positive cells in the intermediate layer of the dysplastic leukoplakias (p=0.0319) and in the basal layer of lesions with poorer evolution (p=0.0133). CONCLUSION: The levels of autophagy increased during the process of oral carcinogenesis and are correlated with poorer behavior of the leukoplakias.

Lepidium meyenii (Maca) does not exert direct androgenic activities.

Maca is the edible root of the Peruvian plant Lepidum meyenii, traditionally employed for its purported aphrodisiac and fertility-enhancing properties. This study aimed at testing the hypothesis that Maca contains testosterone-like compounds, able to bind the human androgen receptor and promote transcription pathways regulated by steroid hormone signaling. Maca extracts (obtained with different solvents: methanol, ethanol, hexane and chloroform) are not able to regulate GRE (glucocorticoid response element) activation. Further experiments are needed to assess which compound, of the several Maca's components, is responsible of the observed in vivo effects.

Olive phenol hydroxytyrosol prevents passive smoking-induced oxidative stress.

BACKGROUND: Oxidative stress is involved in the onset of several degenerative disorders, and epidemiological studies indicate that a high intake of dietary antioxidants, as in the case of the Mediterranean basin, is protective. Olive mill waste waters (OMWWs) are a byproduct of olive oil production rich in phenolic antioxidants, such as hydroxytyrosol. We tested the effects of a low dose of an OMWW extract in a model of sidestream smoke-induced oxidative stress in rats by evaluating the urinary excretion of 8-iso-prostaglandin (PG) F(2alpha) (iPF(2alpha)-III). METHODS AND RESULTS: An OMWW extract (5 mg/kg, providing 414 microg/kg of hydroxytyrosol) was administered to rats daily for 4 days, during which time the animals were exposed to sidestream smoke for 20 minutes once a day. Daily urines were collected, and the urinary excretion of 8-iso-PGF(2alpha) was evaluated as an index of oxidative stress-induced in vivo lipid peroxidation. The exposure of rats to passive smoking increased the urinary excretion of 8-iso-PGF(2alpha) by 44+/-4.2% at 48 hours and by 55+/-10% at 96 hours. Treatment with the OMWW extract was able to completely prevent the increase at 48 hours and resulted in lower increments (34+/-18% versus 55+/-10%) of 8-iso-PGF(2alpha) excretion at 96 hours. CONCLUSIONS: A low dose of hydroxytyrosol, administered through OMWW, reduces the consequences of sidestream smoke-induced oxidative stress in rats.

Diet and prevention of coronary heart disease: the potential role of phytochemicals.

Epidemiological studies, and some clinical trials, demonstrate that a proper diet reduces the rate of occurrence of cardiovascular disorders. Several in vitro studies suggest that some components of plant foods, most of which sharing a phenolic structure, are endowed with interesting 'pharmacological activities'. This article reviews the evidence that links a high dietary intake of phytochemicals from various sources with a reduced incidence of coronary heart disease.

Relevance of dietary glycerophospholipids and sphingolipids to human health.

Glycerophospholipids and sphingolipids participate in a variety of indispensable metabolic, neurological, and intracellular signaling processes. In this didactic paper we review the biological roles of phospholipids and try to unravel the precise nature of their putative healthful activities. We conclude that the biological actions of phospholipids activities potentially be nutraceutically exploited in the adjunct therapy of widely diffused pathologies such as neurodegeneration or the metabolic syndrome. As phospholipids can be recovered from inexpensive sources such as food processing by-products, ad-hoc investigation is warranted.

In vitro differentiation of human monocytes to macrophages results in depletion of antioxidants and increase in n-3 fatty acids levels.

The lipid composition and alpha-tocopherol content of human monocytes were investigated before and after their differentiation to macrophages. The total lipid and protein content per number of cells increased after the differentiation of monocytes by approximately four-fold; a two-fold increase in docosahexaenoic and docosapentaenoic acids and a two-fold decrease in linoleic acid were also noted. As opposed to an initial monocytic vitamin E content of 4.75 pmol/10(6) cells, macrophagic vitamin E levels were undetectable. Changes in vitamin E and fatty acids contents in macrophages, with respect to monocytes, appear to reflect the lipid composition of fetal calf serum, that is low in vitamin E and has a proportionally higher docosahexaenoic acid content than adult human serum.